Process for the obtention of invariant natural killer t cells

Abstract

The present invention pertains to cell culture protocols and particularly provides a method for obtaining Invariant Natural Killer T (iNKT) cells. The method according to the present invention particularly comprises culturing peripheral blood mononuclear cells (PBMC) in a culture medium comprising alpha-Galactosylceramide (alpha-GalCer) and Interleukin 15 (IL-15). The present invention allows for the specific expansion of CD4V iNKT which have a particular interest in the prevention of graft versus host disease in the context of hematopoietic stem cell transplantation.

1 : A method for obtaining Invariant Natural Killer T (iNKT) cells, said method comprising a step of culturing peripheral blood mononuclear cells (PBMC) in a culture medium comprising alpha-Galactosylceramide (alpha-GalCer) and Interleukin 15 (IL-15), wherein the expansion factor of iNKT cells is equal to or higher than 100. 
     
     
         2 : The method according to  claim 1 , wherein the expansion factor of iNKT cells is equal to or higher than 500. 
     
     
         3 : The method according to  claim 1 , wherein said method further comprises a step of screening and isolating CD4 −  iNKT cells. 
     
     
         4 : The method according to  claim 1 , wherein said culturing step is performed over 7 to 21 days, preferably over 14 days. 
     
     
         5 : The method according to  claim 1 , wherein the concentration of IL-15 in the culture medium is comprised between 5 and 15 ng/mL, preferably around 10 ng/mL. 
     
     
         6 : The method according to  claim 1 , wherein the concentration of alpha-GalCer in the culture medium is comprised between 50 and 150 ng/mL, preferably around 100 ng/mL. 
     
     
         7 : The method according to  claim 1 , wherein the PBMCs are introduced in the culture medium at a concentration ranging from 1×10 4  to 1×10 7  cell/mL, preferably from 1×10 5  to 2×10 6  cells/mL. 
     
     
         8 : The method according to  claim 1 , wherein said culture medium further comprises fetal bovine serum (FBS), human AB serum and/or glutamine. 
     
     
         9 : The method according to  claim 8 , wherein the concentration of FBS and/or human AB serum is comprised between 1 and 8%, and the concentration of glutamine is comprised between 1 and 10 mM. 
     
     
         10 : The method according to  claim 1 , wherein said culturing step is conducted at a temperature of 37° C. in presence of 5% of carbon dioxide (CO 2 ). 
     
     
         11 : The method according to  claim 1 , wherein said PBMCs are firstly cultured in presence of alpha-GalCer prior adding IL-15. 
     
     
         12 : The method according to  claim 11 , wherein IL-15 is introduced in the culture medium after 1 to 5 days of culture, preferably after 1 day of culture. 
     
     
         13 : The method according to  claim 1 , wherein said method further comprises a step of replacing 20 to 80% of the culture medium used in the preceding steps with a culture medium comprising IL-15. 
     
     
         14 : The method according to  claim 13 , wherein said step of replacing the culture medium is performed after 6 to 8 days of culture. 
     
     
         15 . (canceled)